Training Manual on DUS Test in Cotton with reference to PPV & FR legislation, 2001

Transcription

1 Training Manual on DUS Test in Cotton with reference to PPV & FR legislation, 2001 Compiled by Dr K Rathinavel, Senior Scientist, Seed Technology Dr S Manickam, Scientist-Sr Scale, Genetics Sh. M Sabesh, Scientist, Computer Applications January, 2005 Citation : Training Manual on DUS Test in Cotton with reference to PPV & FR legislation, 2001, 19 th to 25 th January, 2005 Published by Project Coordinator (Cotton Improvement) and Head All India Coordinated Cotton Improvement Project Central Institute for Cotton Research Regional Station, Coimbatore Year : 2005 DUS testing in Cotton 1

2 Contents: Training Manual on DUS Test in Cotton with reference to PPV & FR legislation, Dus Testing of crop varieties-principles and Procedures - V.A.Amalraj 2. DUS testing of crop varieties- a synthesis on the subject for new PVP-opting countries - S.Mauria 3. Protection of Plant Varieties and Farmers Rights Act-Implementation and Likely Impact on Indian Seed Sector - S.Mauria and S.Manickam 4. Criteria for Plant Variety Protection: Establishment of Dus on the basis of Qualitative and Quantitative Characters - Surendra Prakash 5. Protection of Plant Varieties and Farmers Rights act, 2001 and DUS test- An over view - K.Rathinavel 6. Conduct of DUS Test in Cotton: Planting material, duration, plant population, experimental design, method and observations - K.Rathinavel 7. Testing of varieties for Distinctness, Uniformity and Stability - A. Bharathi 8. Guidelines for DUS Testing In Cotton - Phundan Singh,V.Santhy, P.R.Vijayakumari and R.K.Deshmukh 9. Morphological characterization of Cotton Varieties - S. Manickam 10. Morphological techniques in varietal identification of cotton - A.S. Ponnuswamy and K. Parameswari 11. Physiological and Biochemical techniques for crop variety identification - T.V. Karivaradaraaju 12. Molecular Markers: History, Features and Applications - M. Maheswaran 13. Cotton Varieties in India - Influence of climate, soil and location of cultivation on expression of morphological characters - an overview - K.N. Gururajan 14. Emerging Strategies in Cotton Breeding Programme - K.N.Gururajan, 15. Homogeneity in field Trials - C.S.Praharaj 16. Water and Weed Management in Cotton - P. Nalayini 17. Management of Cotton Diseases in DUS Test Experimental Plots - P. Chidambaram 18. Pest management in Cotton in DUS experimentation - K. Natarajan and T. P. Rajendran 19. Perspectives of Seed Industry under Protection of Plant Varieties and Farmer s Right Act, 2001 and National Seed Policy M.Ramasami DUS testing in Cotton 2

3 Dus Testing of crop varieties-principles and Procedures V.A.Amalraj Pricipal Scientist & Nodal Officer for GR/IPR/DUS testing of Sugarcane Division of Crop Improvement Sugarcane Breeding Institute (ICAR), Coimbatore. Go top Introduction Crop variety improvement research and development based on conventional breeding as well as biotechnological methods (GM varieties) require considerable investments, in terms of both scientific manpower and financial expenditure. Therefore, in order to attract investment and encourage progress and development, plant-breeding programs have to be protected from misuse, and ensure appropriate incentives to the breeders.thus, an effective system of Plant Variety Protection (PVP) will not only be a safeguard against unlawful commercial exploitation of the new varieties but also stimulate the development of new varieties. The International Union for the Protection of New Varieties (UPOV), with 54 member countries ( as in 2004),provides and promotes an effective system of Plant Variety Protection (PVP) with the aim of encouraging development of new plant varieties for the benefit of mankind in member states.india is not yet a member. UPOV has developed guidelines for testing of more than 200 plant species before granting them protection. These Test Guidelines are used as standard reference document world wide in relation to the description of plant varieties and testing of plant varieties to be qualified for Protection in UPOV member states. Plant variety protection General Agreement on Tariffs and Trade (GATT) recognized Agriculture as an enterprise of investment and profit making and included it in the negations for the first time in Uraguay Round ( ). This round led to the establishment of world Trade Organisation (WTO) in 1995 of which India is a signatory. The provisions of the Trade Related Intellectual Property Aspects (TRIPS) of WTO require that plant varieties need protection, either by parenting or by a sui-generics system or by a combination of both (Mauria,2000). The Indian Patents Act of 1970 does not permit patenting of plants or varieties. Patents on plants, considered a strong form of protection, are available in advanced countries like USA, Japan under certain conditions. But in developing countries, patents are replaced by PVP (Plant Variety Protection) in recognition of the fact that variety development involves improvement of already existing ones and not de novo creation. Involves improvement of already existing ones and not de novo creation. Accordingly, India had enacted its own PVP law, Protection of Plant Varieties and Farmers Rights Act in 2001, with provisions for protecting both breeders and farmers rights. This act provides protection of new varieties including extant and farmer s varieties. The grant of plant breeders rights (PBR) entitle the breeder (or his successor, agent, licensee) to exclude others from producing, selling, marketing, distribution, export or import of propagating material of protected varieties for a period of 15 years. The act also permits a breeder to use a protected variety for research purpose. The act allows the farmer to save, sow, resow, exchange, share or sell farm produce including seed of a protected variety. Dus Testing Thus, under the Protection of Plant Varieties and Farmers Rights Act, a new plant variety can be registered and protected for a specific duration; 15 years for annuals and 18 years for vines and trees. Registration and protection can be granted to a variety only if it conforms to the criteria of Distinctness, Uniformity and stability. It means that the new variety has to Distinct- DUS testing in Cotton 3

4 Uniform-Stable (DUS) in its characteristics. This requires the examination of the variety if it conforms to the standards of DUS test. The examination of a variety for DUS generates a description of the Varity, using its relevant characteristics. This examination of a variety is either conducted by the Plant Variety Protection Authority ( Official testing ) or by the breeder seeking protection ( Breeder testing ). In some countries (Japan, New Zealand), both government or official testing and breeder testing are done. Official testing is common in European countries. Under breeder testing (as in USA, Australia), the applicant has to conduct the tests and demonstrate to the PVP examiner that his new variety meets the criteria of distinctness, uniformity and stability. National Test Guidelines The principles and methods on various aspects of DUS testing of new crop varieties have been documented as National Test Guidelines (NTG).NTG has been developed for 35 crops by the National Core Committee constituted by ICAR. NTG contains details on plant material required, conduct of tests, methods and observations, grouping of varieties, characteristics and symbols, table of characteristics, literature and technical questionnaire. Usually the DUS examination requires at least two independent growing cycles. Not less than two centers have been identified for each crop for conduction the tests. Distinct-Uniform-Stable (DUS): Distinct means a variety should be clearly distinguishable by one or more essential characteristics from any other existing variety. The variety is deemed Uniform if it is sufficiently uniform in its relevant characteristics, subject to variation that may be expected from the particular features of its population. The basis of assessment is normally the number of off-types in the variety, judged on the basis of a population standard and an acceptable probability fixed in the corresponding species. To identify off-types in a population, generally visual observation on characteristics may suffice. However, in a few cases/ crops, it may be necessary to make measurements of each plant to apply statistics to decide or not whether a plant is an off-type. In most of the crops, acceptance probability of 95% has been suggested. For vegetatively propagated and self-pollinated varieties, the following standard has been suggested: Sample size Off-types (permissible) < Generally, cross-pollinated varieties exhibit wider variation within the variety. Relative tolerance limits can be found by comparing with comparable varieties. The standard deviation or variance may be used as the criteria for comparison. Recently, UPOV has proposed a statistical method called Combined Over Year Uniformity (COYU) that takes into account variations between years for dealing uniformity in measured (quantitative) characters. The variety is said to be Stable if its relevant characteristics remain unchanged after repeated propagation. Though it is not usually possible to assess stability with in a period of 2 or 3 years, the variety can be considered stable if is shown to be uniform. Characterization The requirement of distinctness, uniformity and stability are assessed on the basis of characteristics. The characteristics are a feature of whole plant or part of plant. Such DUS testing in Cotton 4

5 characteristics may be morphological, biochemical, molecular or any other nature. The table of characteristics chosen by experts forms the main part of test guidelines and of DUS testing. In Genetic resources, the term characteristic is known as descriptors (with descriptor states) and describing a plant based on such descriptor is known as characterization. (Table of characteristics for DUS testing in sugarcane is given in end) a) Morphological characterization: This is based on botanical or morphological descriptors or characters of the plant or plant part. Types of characteristics 1. Qualitative characteristics: Truly qualitative characteristics show discrete discontinuous states and are stable, heritable and uniformly expressed in all environments(ex.shape, Flowercolour, etc) 2. Pseudo-qualitative characteristics: Here, the range of expression is at least partly continuous varying in more than one dimension. In some cases, intermediate states of expression such as weakly expressed are included between absent and strongly expressed. (Ex. Pubescence, Pigmentation, etc.). Qualitative characteristics are assessed visually while quantitative characteristics are usually measured. The following types of assessments are recommended: VG: Visual assessment by a single observation of a group of plants or parts of plant. VS: Visual assessment by observation of individual plants or parts or plants. MG: Measurement by a single observation of a group of plants or parts of plant. MS: Measurement of a number of individual plants or parts or plant. a) Grouping characteristics can be universally used, either individually or collectively, for grouping the similar varieties. These characteristics are considered to be most reliable in distinguishing or discriminating varieties. b) Biochemical characterization: Isozyme based descriptors have been widely used for identification of crop varieties because of their reliability (Smith & Smith 1992, Cooke 1995).UPOV has also included electrophoresis of isoenzymes in maize, soybean, sunflower and of seed proteins in barley, wheat as additional characters for establishing distinctness of varieties. Selection of an appropriate electrophoresis technique provides a potential tool for variety identification, DUS test or grouping of varieties. c) Molecular characterization: Biotechnology has widened the possibilities for applying such technologies to the problem of characterization, varietal identification and protection (Smith, 1995). The two commonly adopted approaches in the use of molecular markers are essentially either probe based such as RFLP (Restriction Fragment Length Polymorphism), or amplification based like RAPD (Random Amplified Polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism), STMS (Sequence Tagged Microsatellites), etc. At present, molecular markers are not being used in DUS testing anywhere, but they are reliable, fast and cost effective to discriminate an EDV (essentially derived variety). Data from these methods may help resolve disputes on identity of germplasm or hybrids (Santhy et al, 2003) DUS test design The use of experimental design with respect to the number of growing cycles, lay out of the trial, number of plants to be examined and method of observation is largely determined by the number and nature of varieties to be examined in a particular trial. In DUS trials, because of the presence of only one treatment factor (variety), the following designs are used DUS testing in Cotton 5

6 1. Completely Randomised Design- if total number of test varieties is small. Several varieties are examined in a number of replications. 2. Randomised complete Block Design- the number of plots per block equals the number of varieties and all varieties are placed in each block. The advantage is that Standard Deviation between plots does not contain variation due to difference in blocks. 3. Randomised incomplete Block Design- in case of large number of varieties. Here, the number of plots per block is less than the number of varieties. In Poland, performed analysis of variance of the results of experiment concerning seven characters in pea varieties showed that randomized complete block and completely randomized designs were more effective than incomplete block (Pilarczyk, 1999). Reference collection To test whether a candidate variety meets the DUS criteria, it is compared with varieties whose existence is a matter of common knowledge. To satisfy the requirement of distinctness, a candidate variety must be clearly distinguishable from all other existing varieties. These varieties are called the Varieties of Common knowledge, which includes: 1) Protected varieties 2) Varieties listed in official register 3) Varieties, subject of an application for protection 4) Varieties listed in any commercial document in which varieties are offered for marketing in its territory as propagating or harvested material, specially where there is no official registration system. 5) Ecotypes and land races 6) Publicly available varieties within plant germplasm collection (genetic resources, old varieties, etc.) Hence, the competent authority before conducing DUS testing, is expected to collect, establish and maintain the collections of these Common knowledge varieties, in the form of viable seeds or of vegetative plant material of varieties. These will form the Reference Collection. Theoretically, varieties in common knowledge have to be considered on a world wide basis and it is necessary to examine DUS criteria in relation to all varieties of common knowledge known worldwide. But, in practice, this can never be realized, as there are limitations in assembling all varieties on a national basis and every nation has to define strategy to produce a National Reference Collection for each crop.therefore, it is obvious that the list of common knowledge varieties for a given species/crop will include a very large number of entries. It is also very important to ensure the authenticity of the collections as well as the source of collections. The issues on setting up and use of reference collections for DUS testing has been given by UPOV (1997) Maintenance of a reference collection of known varieties is essential for efficient DUS testing. For vegetatively propagated species such as rose, potato, sugarcane, though regeneration of varieties is easy, the clones have to be field-maintained in a disease free condition and without loss of vigour. In vitro conservation could be an alternative and serve as a backup collection. In case be an alternative and serve as a backup collection. In case of other crops, viable seeds of reference varieties are placed in cold storage. The quantity of seed of some crops to be stored are given below Crop Seed quantity (g) Crop Seed quantity(g) Cotton variety/hybrid 4000 Groundnut 7000 Cotton parental lines 4000 Pigeon pea 2000 Rice-variety/hybrid 3000 Soybean 3000 Rice-parental lines 1500 Linseed 500 DUS testing in Cotton 6

7 After the establishment of the reference collection, documentation on the passport and characteristics (morphological, isozyme or molecular) is essential. A proper database on the collection has to be created so that searching and identifying varieties most similar to be candidate variety for examination of distinctness is made easy. Wherever necessary, those similar varieties may be grown and directly compared with the candidate variety in case of doubt or dispute. Use of statistical procedures in DUS testing DUS tests are conducted to compare the varieties and the absolute determination of the characteristics. The measurement or observation of the characteristics are analyzed and based on analysis results, decision is taken on DUS criteria. In DUS testing, an experimental unit is a plot with certain plant population. The plot is a subdivision of the field about which the varieties are randomized. Within a plot, the observations are recorded on some characteristics on certain plants in each replication for estimating the variability of the variety. The mean of the individual plant observations in a plot can be considered as the plot measurement for that character. To address the variation of the data within a variety, the following statistical procedures may be adopted 1. Frequency Distribution or Histogram can be used to describe the values of measurement of quantitative characters. 2. Population mean, Variance and Standard deviation: The mean is the sum of all the expressions of the characters in the population divided by total number of observations. The square root of variance is called standard deviation. For quantitative normally distributed characters, the means of two varieties can be calculated. The comparison of two varieties can be made by computing least significant difference (LSD) between two means. If the difference between two means is greater than LSD, the two means are said to be different. 3. Combined-Over-Years-Distinctness (COYD): This is a procedure for computing maximum distance for establishing DUS. For testing of varieties for distinctness based on measured characters, there is a need to establish a minimum distance between varieties. The pair of varieties showing difference greater than the minimum are said to be regarded as Distinct in respect of that character. The method helps in analyzing quantitative data for open pollinated crops where intra-varietal variation occurs. In most countries, tests are conducted in one testing center for 2 or 3 years. For distinctness, the difference between pairs of varieties is tested on character-by-character basis. To determine the minimum difference analysis of variance is used to calculate LSD for comparing variety means. If the over years/locations mean difference between two varieties is greater than the LSD, then the varieties are considered to be distinct in respect of that character. If more consistency between years/location occurs for the position of different varieties, the minimum distance, which is required for assessing distinctness, will be smaller. On the other hand, if there is a strong interaction, the minimum difference will be enlarged. In COYD analysis, the stability of the relative varieties value is taken into account. This combines the information from different environmental conditions rather than considering centers separately. The basic values to be used in the analysis are the annual location variety means. For bulk sampling which gives at least one value for each variety per year / location, it will usually still be possible to use COYD method for distinctness for any degree of bulking as long as at least one value is recorded for each variety in each year/ location and that the bilk samples are representative for the variety. This method replaces a previous method (ANOVA based on individual experiment) in which analysis was year-by-year or location-by-location basis. 4. Combined-Over-Years-Uniformity (COYU): When the uniformity is judged based on measurement; the standard deviation (SD) can be used to summarize the spread of DUS testing in Cotton 7

8 observations. A new variety can then be tested for uniformity by comparing its SD with that of reference variety. However, in some species or crops, varieties with large plants tend to be less uniform than those smaller plants. If the same standard is applied to all varieties then it is possible that some will have to meet very strict criteria while the other face standard, which are easy to satisfy. Above-mentioned problem with SD is addressed by the use of COYU. This procedure adjust the relationship that exists between uniformity, as measured by plantto-plant SD, and the expression of the characteristics, as measured by the variety mean, before setting a standard. The main advantages of COYU are that all varieties can be compared on the same basis and that information from several years of testing may be combines into a single criterion. Statistical software available Many computer softwares for statistical procedures in DUS testing are in use abroad. Few examples of countries and name of programs used in DUS testing are listed in the table below Country Program Name Functions Japan KIRI General Data base software Poland POWT3 POWT5 -Analysis of categorical data -Analysis of variance (for cumulative records) England DUST -Analysis of data from DUS trials Germany SAS-COYD SAS-COYH VERA -Distinctness tests with 1-3 year trial results -Homogeneity test with 2-3 tear trial results -Generates randomized designs for variety trials with up to 225 entries. Denmark SAS -for calculation of data from DUS & VCU trials Netherlands Literature CIS SCIL-image -Oracle database with applications for DUS & VCU trials data storage and analysis -Image analysis package Cooke R.J Review: Gel electrophoresis for the identification of plant varieties. Journal of Chromatography 698: Mauria S DUS testing of crop varieties-a synthesis on the subject for new PVP opting countries.plant varieties and Seeds. 13: Pilarczyk W On efficiency of resolvable incomplete block design in DUS trials on French bean varieties. UPOV TWC/17/2. Santhy V., Mohapatra T., Dadlani M., Sharma S.P. & R.P.Sharma DNA markers for testing distinctness of rice varieties. Plant vars and seeds 13: Smith J.S.C Identification of cultivate varieties by nucleotide analysis. In Identification of food grain varieties, Wrigley C.W. (ed.). AACC, USA. Smith O.S & J.S.C.Smith Measurement of genetic diversity among maize hybrids. A comparison of isozyme, RFLP, pedigree and heterosis data. Maydica 37: UPOV Thoughts on the setting up and use of reference collection for DUS testing. Document TWA/26/5. UPOV Use of statistical procedures in distinctness, uniformity and stability testing. Document TGP/8.5. UPOV The combined over years distinctness and uniformity criteria. Document TWC/18/10. Go top DUS testing in Cotton 8

9 DUS testing of crop varieties- a synthesis on the subject for new PVP-opting countries S.Mauria Crop Science Division, Indian Council of Agricultural Research, Krishi Bhavan, New Delhi, India. Go top The minimum requirement of an effective sui generic system for the protection of plant varieties under the GATT Agreement establishing the WTO points towards the available system under the UPOV. The UPOV Convention provides a system based on DUS testing of crop varieties, accepted and in operation in a large number of countries. A number of new PVPopting countries are also becoming UPOV members to provide the required effective system. Realizing the present-day need for detailed DUS characterization of crop varieties, a synthesis on the subject is attempted. It discusses the criteria for DUS and important technical initiatives in UPOV, besides covering the discussions on new technologies like isozyme electrophoresis and DNA-profiling in DUS testing, the issue of essentially derived varieties, and developments in analysis of DUS data. The Conclusion section presents some essential elements to be considered for developing a DUS/PVP system in new PVP-opting countries. Introduction The question of Plant Variety Protection (PVP) (or Plant Breeders Rights-PBR) was brought into worldwide focus by the Agreement on Trade Related (Aspects of) Intellectual Property Rights (TRIPs), which is the part of the General Agreement on Tariffs and Trade (GATT) establishing the World Trade Organization (WTO) in It is largely a universal agreement with 131 countries being the contracting parties. Article 27.3(b) of the Section on TRIPs in GATT (GATT 1994) provides that (contracting) parties shall provide for the protection of plant varieties either by parents or by an effective sui generic system or any combination thereof. Thus, provision for an effective sui generic system by the contracting parties is the minimum requirement. A legislative framework for PVP is to be provided by the contacting parties in the specified time frame, by 1 January 2000 in developing countries (except least developed countries). The effectiveness of any sui generic system developed by any contracting parties is to be reviewed under the Agreement from November Being signatory to the Agreement, many developing countries, which were not hitherto having any such system, have either already initiated some form of system or are discussing the issue for putting a system in place. Options for a sui generic system have been discussed by Leskien & Flitner (1997) but in the absence of any specified criteria, for judging the effectiveness, mentioned in TRIPs Section of the GATT Agreement, all thinking and developments point toward the system provided by the inter-governmental International Union for Protection of New Varieties of Plants (commonly known as UPOV based on its initials in French - Union Internationale pour la protection des Obtentions Vegetables). UPOV provides a system already accepted and in operation in a large number of countries, and is making efforts to receive recognition under WTO. The Director General of the United Nations World Intellectual Property Organization (WIPO) is the Secretary General of UPOV. The UPOV Convention has two main functions viz. i) it prescribes minimum rights that must be granted to plant breeders by its member States, that is to say, it specifies a minimum scope of protection; and ii) it establishes novelty, distinctness, uniformity and stability, and the requirement of a suitable denomination, as the standard criteria for the grant of protection. The UPOV Convention was signed in Paris in 1961, it entered into force in It was revised in Geneva in 1972, 1978 and The 1978 Act entered into force on 8 November The 1991 Act entered into force on 24 April A concise description of technical criteria for protection is provided in UPOV (1996). DUS testing in Cotton 9

10 Currently, UPOV has 46 member States with only two States i.e. Belgium and Spain bound by the UPOV s 1961 Act (as amended by the Additional Act of 1972), 29 States bound by the 1978 Act and 15 States bound by its latest 1991 Act. Among these, there are only two States from Africa (Kenya, South Africa) and 2 only from Asia (China, Japan). A number of Latin American developing countries have also now become UPOV member now and most of the UPOV member States are in the process of amending their laws to conform to the 1991 Act. Both the 1978 and 1991 Acts have been discussed and debated for implementation in countries which so far are not members of UPOV. One important difference relevant in the present context between the 1978 and 1991 Acts is that, in the UPOV (1978) Act, a protected variety can be modified in a very limited respect e.g. by reselection, mutation, the addition of a gene etc. and provided variety it can be separately protected without any obligation to the breeder of the protected variety. The UPOV (1991) Act provides that varieties that are essentially derived from a protected variety in this way can still be protected but cannot be marked without the permission of the breeder of the protected variety from which they are derived. Varieties are essentially derived for this purpose only when they are virtually entirely constructed upon the basis of the protected varieties from which they are derived. Discussions are also alive on the issue of unprotected varieties already in public domain. Realizing that the UPOV (1991) Act will ultimately come into force to have its impact on global agriculture, many developing countries like India, and countries of the Andes region (Seiler, 1998), also included this clauses in their draft legislations considering their own strengths in variety development, testing and commercial release. Obviously, the requirements of Distinctness, Uniformity and Stability (commonly referred to as DUS or sometimes as DHS, wherein Uniformity is substituted as Homogeneity ) under UPOV to grant PVP have to be first understood on extant crop varieties of a country. This is necessary because for granting PVP certificates to new varieties, consideration has to be given to obsolete varieties no more in trade as they are of common knowledge, even if they are known by incomplete and imprecise description in literature. Besides, the criteria of common knowledge in granting a PVP certificate cannot be limited to national borders, and similar varieties known anywhere in the world may have to be taken into account. In a new PVP-opting country, its extant varieties can thus initially serve as basic materials to study DUS followed by establishing DUS in new crop varieties alongside the references developed from extant varieties. These extant varieties can thus serve as example varieties, for comparison for character states of particular characteristics, and for studying DUS on new crop varieties. In DUS testing, the principal test is of Distinctness, and detailed examination of diagnostic characteristics of crop varieties and their parental lines has become imperative, to establish this requirement. With the above background, the following is an effort to synthesize at one place the available information on DUS testing of crop varieties, which may be helpful particularly for the new PVP-opting countries where DUS testing systems would have to be established ab initio. It includes the important discussions in UPOV, which is playing an important role in assisting its member States and new PVP-opting countries in their endeavor of developing appropriate systems for meeting the requirements. It needs to be noted that while there may not be much difficulty in establishing a system de nova in any new PVP-opting country by largely adopting from already established systems in many developed countries, there are still many unresolved issues, which UPOV continues to deliberate upon to continually guide its member States and other interested countries / organizations. The author s personal understanding, developed while attempting DUS testing in India for the first time following UPOV Test Guidelines using Indian public maize inbred, was the incentive for attempting this synthesis. DUS testing in Cotton 10

11 Need for detailed examination of Diagnostic characteristics of crop varieties In early days, all over the world, a small list of descriptors was sufficient to distinguish between crop varieties in use. However, in the recent decades, the world witnessed the emergence of large and highly competitive variety development programmes, particularly in the developed programmes, particularly in the developed countries and in some of the developing countries. At the global level, a large number of new candidate varieties are being generated for testing every year, thus underlining the need for establishing their clear-cut diagnostic features. The technology-rich developed countries had obviously realized this requirement much earlier and had, accordingly, tuned their systems to meet the requirement. In most of the developing countries now considering implementing a PVP-system, while certain diagnostic features for released crop varieties are generally known and followed in seed certification procedures, accurate identification keys, giving detailed description on a comparative basis with clear-cut features of distinctness are, in general, lacking; and thus cases of confusion in seed certification and quality control, if such systems are existing, are also not uncommon. The example of India, who benefited greatly from the Green Revolution is cited and compared here. The country has an established system on variety development, testing and release; and over 2600 crop varieties are already notified for commercial cultivation. In India, the variety testing and release system, undertaken through a crop commodityspecific coordinated varietal evaluation system with a large network of cooperating centers in public and private sectors, basically concentrates on generating data on parameters like yield, quality, reaction to important diseases and pests under field conditions and artificial epiphytotics, performance under different agronomic management schedules etc.(tandon, 1992). It is thus more akin to a VCU (Value for Cultivation and Use) test in the European Economic Community (EEC) countries aimed at stimulating plant breeders to produce varieties, which are an improvement over existing varieties (Bould, 1992) done by the Government-designated authorities. The latter test requiring a detailed botanical examination using a standard list of descriptors and is intended to remove any confusion in naming new varieties, which was a major problem in EEC countries in earlier times. Besides meeting the requirement of registration of the new variety, the botanical description is also used for awarding PVP, for which uniqueness is an essential requirement. The need for a detailed examination of diagnostic characteristics thus becomes imperative in new PVP-opting countries to maintain identity of released and notified varieties and their parental lines. With respect to India, Sharma (1991) has indicated that no system of variety registration exists in India apart from the rather vague variety release proposals provided by the breeders and there is a need for establishing a DUS testing system. Such work additionally assists in protecting morphologically, and often agronomically, similar but distinct varieties when a PVP system is established de novo in a country. The requirement also finds support from the work of Singhal & Prakash (1992) who have identified a high degree of resemblance in morphology in recently developed wheat varieties in India. Virk & Witcombe (1998) have also stated that the selection strategy employed in the All India Coordinated Crop Improvement Programmes, the system of multi-locational testing of new varieties, concentrates on selection for yield with emphasis in selection on one important adaptive trait i.e. flowering time, towards which there is strong stabilizing selection. A similar, or even less intensive, system is the general phenomenon in most of the developing countries. In the USA, the entire responsibility for identity of seed material and DUS testing rests with the breeder, and the records provided by the breeders are maintained in PVP offices providing PVP-certificates. In Australia, New Zealand and a few other countries, systems marginally different than of EEC countries are in existence. Also, the technical criteria for the grant of rights differs from one country to another and even the variety concept is not seen in the same light in all the countries. The technical standards DUS testing in Cotton 11

12 and testing procedures likewise depend largely on the expertise of the officials concerned. This lack of harmonization has caused problems, especially when a breeder sought protection for his variety in several countries. Obviously, the developed countries and their private seed sector considered availability of effective PVP systems as necessary in developing countries in order to safeguard the interest of plant breeders as well as to play an important role in the global agriculture. This was made possible by getting included the requirement of an effecting systems for plant variety protection through the multilateral negotiations under GATT. As contracting parties in WTO, and recognizing current efforts by UPOV for harmonization of procedures, a general description of UPOV s criteria is first provided for such new PVP-opting countries. UPOV s criteria for Distinctness Uniformity and Stability A technical examination performed according to standardized principles established by UPOV comprises a comparative growing trial which involves sampling, observation and measurement, processing and evaluation. These trials are conducted either by the national government authorities themselves or on their behalf by specialized bodies or, to varying degrees by the applicants or breeders themselves. In UPOV, the crop-specific test guidelines for conduct of DUS tests are supported by a general introduction to these test guidelines (UPOV, 1979). This general introduction to crop-specific test guidelines is presently under revision (UPOV, 1999a), and is likely to be oriented for guidelines on DUS testing procedures as a whole rather than just supporting the crop-specific Test Guidelines, thereby also aiming to provide guidance on DUS testing where there are no UPOV Test Guidelines available (e.g. for new species).nevertheless, the majority of the points on conduct of DUS tests are well-accepted principles. Accordingly, a brief introduction on UPOV s criteria for Distinctness, Uniformity and Stability is synthesized here to serve the immediate requirement of new PVP-opting countries. Distinctness According to Article 7 of UPOV (1991) Act, a variety shall be deemed distinct if it is early distinguishable from any other variety whose existence is a matter of common knowledge at the time of the filling of the application. Two varieties have to be considered distinct if the difference i)has been determined at least in one testing place, ii)is consistant. In the case of true qualitative characteristics, two distinct varieties show expression, which fall into two different states. In the case of other qualitative handled characteristics (i.e. some visually assessed quantitative characteristics), a degree of continuity has to be taken into account in establishing distinctness, and thus a different character state not be sufficient to establish distinctness. When distinctness depends on measured characteristics, the difference has to be considered clear if it occurs with one percent probability of an error, for example, on the basis of the method of the Least Significant Difference (LSD). The differences are consistent if they occur with the same sign in two out of three growing seasons. In order to take into account the variation between years, UPOV has developed a more sophisticated method, the Combined Over Years Distinctness (COYD) method. It is supplemented by a further LSD method for cases where certain standards required for the COYD analysis cannot be met. Its main use is for measurements in cross-fertilized varieties, but, if so desired, it can also be used for measurements in vegetatively propagated or self-fertilized varieties. If a normally visually assessed quantitative characteristic is the only distinguishing characteristic in relation to another variety, it should be measured, in case of doubt, if this is possible with reasonable effort. A direct comparison between two similar varieties is advised since pair-wise comparisons show the least bias. Distinctness can be established if consistent differences (significant differences with the same sign) are found in pair-wise comparisons, but if DUS testing in Cotton 12

13 they can be expected to recur in the following trials. The number of comparisons has to be sufficient to allow a comparable reliability as in the case of measured characteristics. In some cases, differences between two varieties may be observed in several separately assessed characteristics but the establishment of distinctness depends on using a combination of such data. Currently, combined characteristics may only be used where they have clear biological meaning and the degree of reliability is comparable with that provided for measured or normally visually observed characteristics. In identification of characteristics for establishing distinctness, UPOV has obviously put those characteristics, in individual Test Guidelines, on which sufficient knowledge and experience has been gained. Nevertheless, it is also indicated that the tables of characteristics provided in the Test Guidelines are not exhaustive and may be enlarged by further characteristics if this proves to be useful. Such characteristics should obviously have discriminating properties. Different degrees of uniformity are not accepted as a characteristic for distinctness. It must also be mentioned here that the crop-specific Test Guidelines are harmonized for making descriptions and include a list of characteristics, and their character states, which are indicated by different numerical Notes on a 1-9 scale. For the decision on distinctness, uniformity and stability, these guidelines only represent the first step. The Test Guidelines are silent on the minimum distance (discussed later) required in each characteristic and thus a decision on distinctness can never be based on the description resulting from the Test Guidelines. However, to make the first step meaningful and allow a first idea on the possibility of distinction, the following is advised to be observed. i. In true qualitative characteristics, each character state is clearly separated from the other without any transition; the minimum distance is therefore always one Note. There are, however, only very few true qualitative characteristics. ii. In quantitative characteristics which are observed visually, it should be aimed at setting up a scale of states- if possible as a rule with a difference of two Notes- which could lead to a clear difference ( this is meant by the requirement that the states be meaningful). However, these two Notes are no absolute standard for the minimum distance. Depending on the testing place, the year or other environmental conditions, variety collection or special pair of varieties, the minimum distance may be more or less than two Notes, e.g. three, four or five Notes in a characteristic affected to a larger degree by the environment; or may be one only or even inside one Note, distinction may be possible. It is up to the expert doing the observations to take the necessary precautions. The variety description based on the Test guidelines should therefore never be used alone for the decision on distinctness and a general yardstick of two Notes is only an aim for the experts who draft the Test Guidelines but never for the user. iii. Characteristics which are handled like qualitative characteristics, but which are not really qualitative characteristics, should be handled in such a way that possible fluctuations are taken into account when distinctness is assessed. Therefore, one cannot automatically presume that the minimum distance is one Note. The sequence of the states should in such characteristics rather be chosen in such a way that as a rule a minimum distance is two Notes could be expected. Accordingly, the states may be for instance for growth habit; erect (1), semi-erect (2), prostrate (3) in one species; and erect (1), semi-erect (3), intermediate (5), semi-prostrate (7), prostrate (9) in another species; and for a third species the states may be set up again in a different way. The same reservations as for quantitative characteristics apply, however, and the description based on the Test Guidelines should not be used alone to take a decision on distinctness. In practice, some countries regard consecutive states of true qualitative characteristics to be distinct (1 & 2), while only every second state of a quantitative characteristics is regarded as distinct (1 & 3, 2 & 4). The majority of the UPOV member States do not follow this idea. There is a frequent misinterpretation of the use of the Test Guidelines, which may stem from the title of the DUS testing in Cotton 13

14 Test Guidelines. The function of the Test Guidelines is mainly for description purposes. Experts have to be avoiding mixing description and distinction of a variety. It is possible that two samples of plant material could have different descriptions but is not sufficiently distinct to be from two varieties eligible for protection. Therefore, the yardstick of two states of expression in quantitative characteristics is for the drafter of the Test Guidelines and not for the user. Test Guidelines are, as stated, merely guidelines and not instructions for the testing at a certain place. Uniformity (Homogeneity) According to Article 8 of UPOV (1991) Act, the variety shall be deemed to be uniform if, subject to the variation that may be expected from the particular features of its propagation, it is sufficiently uniform in its relevant characteristics. In effect, t means that variation shown by a variety, depending upon its breeding system, must be as limited as necessary to permit accurate description and assessment of distinctness and to ensure stability. This requires a certain tolerance, which will differ according to the reproductive system of the variety. The approaches to vegetatively propagated varieties, truly self-pollinated varieties, mainly self-pollinated varieties; cross-pollinated varieties, synthetic varieties and hybrid varieties are thus necessarily very different. In case of vegetatively propagated and truly self-pollinated varieties, the maximum acceptable number of off-types in samples of various sizes is specifically provided in the Test Guidelines. For mainly self-pollinated varieties, a higher tolerance is admitted and the populated standard for the calculation of the maximum number of off-types allowed for vegetatively propagated and truly self-pollinated varieties is generally doubled. In the case of cross-pollinated varieties, including synthetic varieties, which normally exhibit wider variations within the variety, relative tolerance limits are used through comparisons with comparable varieties already known. For measured characteristics, a variety is considered not to be homogeneous in the measured characteristics concerned, if its variance exceeds 1.6 times the average of the varieties used for comparison (UPOV, 1979). In order to take into account variations between years, the Combined Over Years Uniformity (COYU) method has been developed, which is a further development of the earlier mentioned COYD method used for distinctness. In the case of visually assessed characteristics, the number of plants visually different from those of the variety should not significantly (5% probability of an error) exceed the number found in comparable varieties already known. Single cross hybrid varieties have to be treated as mainly self-pollinated varieties, but an additional tolerance has to be allowed for inbred plants. It is not possible to fix a percentage as the decisions differ according to the species and the breeding method. The maximum number of off-types tolerated is proposed to be fixed and provide in the Test Guidelines of that crop. For other categories of hybrids, a segregation of certain characteristics is acceptable if it is in agreement with the formula of the variety. If the heredity of a clear-cut segregating characteristics is known, this characteristic has to be treated as a qualitative characteristic. If the described characteristic is not clear-cut, it has to be handled as in the case of other kinds of characteristics of cross-pollinated species with high-inbred depression or non-uniform parent lines, only relative uniformity standards are advised to be applied. It is for the national authorities to take the decision where the parent did not show uniformity and it follows from this that the formula of the hybrid must be known to the testing authority. In individual crops Test Guidelines, parameters like sample size, population standard, acceptance probability and maximum number of off-types permitted are also indicated to meet the requirements of Uniformity. Any plant is to be considered an off-type if it differs in the expression of any characteristic, of the whole plant or part of the plant, from that of the variety, taking into consideration the particular species. An admixture is considered an off-type. However, plants that are very different from the variety may be disregarded as long as their number does DUS testing in Cotton 14

15 not interfere with the test (dependent on the judgment of the expert). An interim period for adjustment to adoption of Test Guidelines is also provided for new PVP-opting countries. Stability According to Article 9 of UPOV (1991) Act, a variety shall be deemed stable if its relevant characteristics remain unchanged after repeated propagation or, in the case of particular cycles of propagation, at the end of each such cycle. It is not generally possible during a period of two to three years (testing time required) to perform tests on stability which lead to the same degree of certainty as the testing of distinctness and homogeneity. Generally, when a submitted sample has been shown to be homogenous, the material can also be considered stable. Nevertheless, during the testing for distinctness and homogeneity, careful attention has to be paid to stability. As far as necessary, stability has to be tested by growing a further generation or new seed stock to verify that it exhibits the same characteristics as those shown by the previous material supplied. Important Technical Initiatives in UPOV UPOV is continually striving to harmonize the systems developed in individual countries. Thiele-Wittig (1992) reviewed the development of technical work is handled by five Technical Working in UPOV. In UPOV, the technical work is handled by five Technical Working Parties, four on different crop-commodity groups (viz.agricultural crops, fruit crops, ornamental plants and forest trees, and vegetables), and one for automation and computer programs (TWC). Besides, there is a Working Group on Biochemical and Molecular Techniques and DNA-profiling in particular (BMT). The Council of UPOV has appointed a Technical Committee to which the Working Parties report. UPOV s main achievements in the technical field cover important topics viz. common understanding on basic principles for the testing of new varieties, harmonized and adopted individual test procedures on about 170 genera or species, cooperation between member States and distribution of tasks, cooperation with applicants and breeders in the growing tests, discussion on use of new technologies and data analysis methods including on new types of characteristics like electrophoresis, colour measurements, image analysis etc. The general introduction to the test guidelines and crop-specific guidelines on conduct of DUS tests provide the basic information. Crop-specific test guidelines mainly cover the traditional morphological and physiological characteristics studied in field testing of materials. Other characteristics obtained with the help of new, generally laboratory-based technologies, which have recently been considered in a few species, have to fulfill the same requirements as other traditional characteristics before being accepted for DUS testing. There should be an accepted standardized method for observation of the characteristic and it should lead to reliable and repeatable results. Important subjects discussed by TWC include inventory of databases and their structure, inventory of existing hardware, inventory of data processing functions applied and/ or required in the plant variety protection offices, methodology for the testing of homogeneity in cross-fertilized and self-fertilized crops, evaluation of combined over years analysis for distinctness and uniformity testing, and content and format of descriptions of varieties to facilitate automation. Information from UPOV could hitherto be obtained only on paper, but initiatives have begun in the recent past for computerization of the information bank available with UPOV and its member States. DUS testing in Cotton 15